RELATED APPLICATIONS
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with Government support under NIH R37DE013033 awarded by
the National Institutes of Health and MRSEC DMR-0820484 awarded by the National Science
Foundation. The Government has certain rights in the invention.
FILED OF THE INVENTION
[0003] This invention relates to biocompatible hydrogel compositions.
BACKGROUND OF THE INVENTION
[0004] Over the recent decades, biocompatible polymers have been used to form scaffolds
that act as carriers for cell transplantation, or to recruit host cell populations
into the device.
SUMMARY OF THE INVENTION
[0005] The invention provides compositions and methods to form porous hydrogels. For example,
pores are formed in situ within hydrogels following hydrogel injection into a subject.
Pores that are formed in situ via degradation of sacrificial porogens within the surrounding
hydrogel (bulk hydrogel) facilitate recruitment or release of cells. For example,
the resulting pore is within 5% of the size of the initial porogen.
[0006] Disclosed herein is a material that is not initially porous, but which becomes macroporous
over time resident in the body of a recipient animal such as a mammalian subject.
These compositions are associated with significant advantages over earlier scaffold
compositions. The hydrogels described herein are well-suited to initially protect
transplanted cells from host inflammatory responses, and then release transplanted
cells after inflammation has subsided (e.g., after 12 hours, or 1, 3, 5, 7, or 10
days or more post-transplantation, i.e. resident in the body of the recipient). The
hydrogels described herein also double as a surgical bulking agent, further minimizing
inflammation in the host, and then later releasing cells.
[0007] Accordingly, the invention provides a composition as defined in the appended claims.
The composition comprises a porogen hydrogel and a bulk hydrogel, which composition
is not initially macroporous and becomes macroporous over time when resident in the
body of a receipient, wherein the porogen hydrogel degrades at least 10% faster (e.g.
, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%,
at least 45%, or at least 50% faster) than the bulk hydrogel following residence in
said subject, leaving macropores having a diameter of greater than 20 µm in its place,
and wherein (a) said porogen hydrogel comprises oxidized alginate; or (b) said porogen
hydrogel comprises a shorter polymer than said bulk hydrogel. For example, the first
hydrogel is a porogen and the resulting pore after degradation insitu is within 25%
of the size of the initial porogen, e.g., within 20%, within 15%, or within 10% of
the size of the initial porogen. Preferably, the resulting pore is within 5% of the
size of the initial porogen. The porogen hydrogel degrades more rapidly than the bulk
hydrogel, because the porogen hydrogel is more soluble in water (comprises a lower
solubility index). Alternatively, the porogen hydrogel degrades more rapidly because
it is cross- linked to protease-mediated degradation motifs as described in USSN
10/980,989 to Zilla.
[0008] The molecular mass of the polymers used to form the porogen hydrogel composition
are approximately 50 kilodaltons (kDa), and the molecular mass of the polymers used
to form the bulk hydrogel composition comprises approximately 250 kDa. A shorter polymer
(e.g. that of a porogen) degrades more quickly compared to that of a longer polymer
(e.g., that of the bulk composition). Alternatively, a composition is modified to
render it more hydrolytically degradable by virtue of the presence of sugar groups
(e.g., approximately 3-10% sugar of an alginate composition). In another example,
the porogen hydrogel is more enzymatically degradable compared to the bulk hydrogel.
The composite (first and second hydrogel) composition is permeable to bodily fluids,
e.g., such as enzyme which gain access to the composition to degrade the porogen hydrogel.
In some cases, the second hydrogel is cross- linked around the first hydrogel, i.e.,
the porogens (first hydrogel) are completely physically entrapped in the bulk (second)
hydrogel.
[0009] Cells or bioactive factors (e.g., growth factors such as granulocyte/macrophage colony
stimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), condensed
oligonucleotides, e.g., CpG, or plasmid DNA) are optionally encapsulated either into
the porogen phase, bulk hydrogel phase, or into both phases. The porogens degrade
in situ over a time-course pre-determined by the user. Upon degradation of the porogens,
cells are released from or may migrate into the material. However, because they initially
lack pores, pore-forming hydrogels are useful to provide mechanical support immediately
after formation. Suitable bioactive factors include vascular endothelial growth factor
(e.g., VEGFA; GenBank Accession Number: (aa) AAA35789.1 (GI:181971), (na) NM_001171630.1
(GI:284172472)), acidic fibroblast growth factor (aFGF, Genbank Accession Number:
(aa) AAB29057.2 (GI: 13236891), (na) NM_000800.3 (GI:222144219)), basic fibroblast
growth factor (bFGF; GenBank Accession Number: (aa) AAB21432.2 (GI:8250666), (na)
A32848.1 (GI:23957592)), placenta growth factor (PIGF or PLGF; GenBank Accession Number:
(aa) AAH07789.1 (GI:14043631), (na) NM_002632.4 (GI:56676307)), leptin (Genbank Accession
Number: (aa) CBI71013.1 (GI:285310289), (na) NM_000230.2 (GI:169790920)), hematopoietic
growth factor (e.g., HGF, Genbank Accession Number: (aa) AAA64297.1 (GI:337938), (na)
NM_000601.4 (GI:58533168)), VEGF receptor-1 (VEGFR-1, Genbank Accession Number: (aa)
NP_002010.2 (GI:156104876)), VEGFR-2 (Genbank Accession Number: (aa) AAC16450.1 (GI:3132833),
(na) EU826563.1 (GI:194318421)), transforming growth factor-β (TGF-β, Genbank Accession
Number: (aa) AAA36738.1 (GI:339564), (na) NM_000660.4 (GI:260655621)), bone morphogenetic
protein (e.g. , BMP-4, Genbank Accession Number: (aa) NP_570912.2 (GI:157276597),
(na) NM_001202.3 (GI: 157276592)), insulin-like growth factor (IGF-1, Genbank Accession
Number: (aa) CAA01954.1 (GI:1247519), (na) NM_001111283.1 (GI: 163659898)), fibroblast
growth factor-2 (FGF-2), platelet-derived growth factor (PDGF; GenBank Accession Number:
(aa) AAA60552.1 (GI:338209), (na) NM_033023.4 (GI:197333759)), epidermal growth factor
(EGF, Genbank Accession Number: (aa) AAH93731.1 (GI:62740195)), transforming growth
factor-a (TGF-a, Genbank Accession Number: (na) NM_003236.2 (GI:153791671)), nerve
growth factor (NGF, Genbank Accession Number: (aa) AAH32517.2 (GI:34192369), (na)
NM_002506.2 (GI:70995318)), brain-derived neurotrophic factor (BDNF, Genbank Accession
Number: (aa) CAA62632.1 (GI:987872), (na) NM_170731.4 (GI:219842281)), neurotrophin-3
(NT-3, Genbank Accession Number: (aa) NP_001096124.1 (GI:156630995), (na) NM_001102654.1
(GI:156630994)), ciliary neurotrophic factor (CNTF, Genbank Accession Number: (aa)
AAB31818.1 (GI:633830), (na) NM_000614.3 (GI:209574322)), and glial cell line- derived
neurotrophic factor (GDNF, Genbank Accession Number: (aa) CAG46721.1 (GI:49456801),
(na) NM_000514.3 (GL299473777)). Other suitable factors include anti-VEGF antibody,
anti-aFGF antibody, anti-bFGF antibody, anti-PIGF antibody, anti-leptin antibody,
anti-HGF antibody, anti-VEGFR-1 antibody, anti-VEGFR-2 antibody, batimastat (BB-94),
marimastat (BB-2516), thalidomide, O-(chloroacetylcarbamoyl)-fumagillol (TNP-470),
carboxyamidotriazole (CAI), mitoxantrone, doxorubicin, SU5416, anti-TGF-β antibody,
anti-BMP antibody, anti-IGF-1 antibody, anti-FGF-2 antibody, anti-PDGF antibody, anti-EGF
antibody, anti-TGF-a antibody, and anti-VEGF antibody. Other bioactive factors suitable
for encapsulation either into the porogen phase, bulk hydrogel phase, or into both
phases include FMS-like tyrosine kinase 3 ligand (Flt3 ligand; Genbank Accession Number:
(aa) AAI44040 (GI:219519004), (na) NM_004119 (GI: GI: 121114303)), anti-flt3 ligand,
hepatocyte growth factor (Genbank Accession Number: (aa) AAB20169 (GI:237997)), and
stromal derived factor 1 (SDF-1).
[0010] Alternatively, an adenovirus is optionally encapsulated either into the porogen phase,
bulk hydrogel phase, or into both phases. For example, the adenovirus encodes runt-related
transcription factor (e.g., Runx2; Genbank Accession Number: (aa) CAI13532 (GI:55959066),
(na) NM_001024630 (GI:226442782)), a key transcription factor associated with osteoblast
differentiation. In another aspect, the adenovirus encodes MyoD (Genbank Accession
Number: (aa) CAA40000 (GI:34862), (na) NM_002478 (GI:77695919)), a protein with a
key role in regulating muscle differentiation. Alternatively, the adenovirus encodes
bone morphogenetic protein, e.g., BMP-2 (Genbank Accession Number: (aa) AF040249_1
(GI:6649952), (na) NM_001200 (GI:80861484)) or BMP-4 (Genbank Accession Number: (aa)
NP_570912.2 (GI: 157276597), (na) NM_001202.3 (GI: 157276592) reference). BMP-2 is
involved in,
inter alia, bone repair, while BMP-4 is involved in the repair of cardiac tissue. In one aspect,
an adenovirus that encodes Runx and an adenovirus that encodes BMP-2 are encapsulated
into the hydrogel.
[0011] Cells suitable for being encapsulated either into the porogen phase, bulk hydrogel
phase, or into both phases include mesenchymal stem cells, myoblasts, vascular progenitor
cells {
e.g., an outgrowth endothelial cell), differentiated cells derived from embryonic stem
cells or induced pluripotent stem cells, induced pluripotent cells, or cells that
were directly reprogrammed from a fibroblast to a differentiated state.
[0012] In some examples, the porogen composition comprises cells, and in other examples,
the bulk composition comprises cells. If cells are present in the composition {
e.g., having been seeded during fabrication), the cells are deployed out of the composition
after administration into a mammalian subject. Alternatively, the composition does
not comprise cells; however, upon administration into tissues of a mammalian subject
{e.g., implantation into a human patient), cells are recruited into the composition.
The mammal can be any mammal, e.g., a human, a primate, a mouse, a rat, a dog, a cat,
a horse, as well as livestock or animals grown for food consumption, e.g., cattle,
sheep, pigs, chickens, and goats. In a preferred embodiment, the mammal is a human.
Alternatively, the subject can be a non-mammalian animal such as xenopus, salamander,
or newt.
[0013] The invention provides methods of deploying cells from a scaffold into tissues of
a mammalian subject, comprising administering to a subject a composition comprising
a first hydrogel and a second hydrogel, wherein the first hydrogel degrades at least
10% faster than the second hydrogel, and wherein the composition lacks pores at the
time of administration, and wherein the composition comprises pores following residence
in said subject, and wherein the first hydrogel or the second hydrogel comprises an
isolated cell.
[0014] A methods of recruiting cells into a scaffold in vivo is carried out by administering
to a subject a composition comprising a first hydrogel and a second hydrogel, wherein
the first hydrogel degrades at least 10% faster than the second hydrogel and wherein
the composition lacks pores at the time of administration, but comprises pores following
residence in the subject. For example, pores are created due to the relative degradability
or solubility of a first hydrogel composition compared to a second hydrogel composition,
e.g., a porogen composition compared to a bulk composition.
[0015] Porosity influences recruitment and/or egress of the cells from the composition.
Pores are nanoporous, microporous, or macroporous. For example, the diameter of nanopores
is less than about 10 nm. Micropores are in the range of about 100 nm to about 20
µm in diameter. Macropores are greater than about 20 µm (
e.g., greater than about 100 µm or greater than about 400 µm). Exemplary macropore sizes
include 50 µm, 100 µm, 150 µm, 200 µm, 250 µm, 300 µm, 350 µm, 400 µm, 450 µm, 500
µm, 550 µm, and 600 µm. Macropores are those of a size that permit a eukaryotic cell
to traverse into or out of the composition. In one example, a macroporous composition
has pores of about 400 µm to 500 µm in diameter. The preferred pore size depends on
the application. For example, for cell deployment and cell release, the preferred
pore diameter is greater than 50 µm.
[0016] The size of the porogen is related to the size of the overall composite material.
For example, for the material to stay intact, the porogen diameter is < 10% of the
smallest dimension of the overall composite. The density of porogens is between 10-80
percent of the overall volume of the composite composition. For example, the density
of porogen is between 15% and 75%, between 20% and 70%, between 25% and 65%, between
30% and 60%, or between 35% and 55% of the overall volume. Preferably, the density
of porogens is at least 50% of the overall volume to achieve optimal cell recruitment
to the hydrogel or cell release from the hydrogel.
[0017] The hydrogel has an elastic modulus of between about 10 to about 1,000,000 Pascals
(
e.g., from about 10 to about 100,000 Pa, from about 10 to about 150,000 Pa, from about
10 to about 200,000 Pa, from about 10 to about 300,000 Pa, from about 10 to about
400,000 Pa, from about 10 to about 500,000 Pa, from about 10 to about 600,000 Pa,
from about 10 to about 700,000 Pa, from about 10 to about 800,000 Pa, or from about
10 to about 900,000 Pa). Preferably, the slowly-degrading hydrogel comprises an elastic
modulus of about 20 kilo Pa to 60 kPa,
e.g., 25 kPa to 55 kPa, 30 kPa to 50 kPa, or 35 kPa to 45 kPa. The rapidly-degrading hydrogel
comprises an elastic modulus of at least 40 kPa initially in order to maintain integrity
during encapsulation prior to degradation.
[0018] Preferably, the slowly-degrading hydrogel (
i.e., the second hydrogel or "bulk") comprises high molecular weight peptides with an amino
acid sequence of RGD which mimic cell adhesion proteins. Alternatively, the slowly-degrading
hydrogel comprises a different adhesive peptide amino acid motif such as PHSRN or
DGEA. For example, the slowly-degrading hydrogels are preferably modified with 2-10
RGD peptides / polymer (
e.g., alginate polymer).
[0019] By "hydrogel" is meant a composition comprising polymer chains that are hydrophilic.
Exemplary hydrogels are comprised of materials that are compatible with cell encapsulation
such as alginate, polyethylene glycol (PEG), PEG-acrylate, agarose, and synthetic
protein (
e.g., collagen or engineered proteins (
i.e., self-assembly peptide-based hydrogels). For example, a commercially available hydrogel
includes BD™ PuraMatrix™. BD™ PuraMatrix™ Peptide Hydrogel is a synthetic matrix that
is used to create defined three dimensional (3D) micro-environments for cell culture.
[0020] For example, the hydrogel is a biocompatible polymer matrix that is biodegradable
in whole or in part. Examples of materials which can form hydrogels include alginates
and alginate derivatives, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic
acid) (PLGA) polymers, gelatin, collagen, agarose, natural and synthetic polysaccharides,
polyamino acids such as polypeptides particularly poly(lysine), polyesters such as
polyhydroxybutyrate and poly-epsilon.-caprolactone, polyanhydrides; polyphosphazines,
poly(vinyl alcohols), poly(alkylene oxides) particularly poly(ethylene oxides), poly(allylamines)(PAM),
poly(acrylates), modified styrene polymers such as poly(4-aminomethylstyrene), pluronic
polyols, polyoxamers, poly(uronic acids), poly(vinylpyrrolidone), and copolymers of
the above, including graft copolymers. Synthetic polymers and naturally-occurring
polymers such as, but not limited to, collagen, fibrin, hyaluronic acid, agarose,
and laminin-rich gels may also be used.
[0021] A preferred material for the hydrogel is alginate or modified alginate material.
Alginate molecules are comprised of (1-4)-linked β-D-mannuronic acid (M units) and
α L-guluronic acid (G units) monomers, which can vary in proportion and sequential
distribution along the polymer chain. Alginate polysaccharides are polyelectrolyte
systems which have a strong affinity for divalent cations (
e.g., Ca
+2, Mg
+2, Ba
+2) and form stable hydrogels when exposed to these molecules.
[0022] The compositions described herein are suitable for clinical use,
e.g., bone repair, regeneration, or formation; muscle repair, regeneration, or formation;
and dermal repair, regeneration, or formation. For example, the compositions are applied
to bone fractures alone or together with a bone adhesive (cement) or glue or to diseased
or injured muscle tissue. The hydrogels (seeded with cells or without cells) are injected
at the site of disease, injury, or fracture (in the case of bone or cartilage). For
example, the hydrogels are injected into or onto bone. Exemplary bioactive factors
for use in promoting bone or cartilage repair, regeneration, or formation include
BMP-2, BMP-4, or RunX.
[0023] In some cases, the composition recruits cells to promote bone or cartilage repair,
regeneration, or formation. Alternatively, the first hydrogel or the second hydrogel
comprises an isolated bone cell selected from the group consisting of an osteoblast,
an osteocyte, an osteoclast, and an osteoprogenitor. Alternatively, the first hydrogel
or the second hydrogel comprises an isolated cartilage cell, wherein the isolated
cartilage cell comprises a chondroblast. The isolated bone cell or an isolated cartilage
cell is an autologous cell or an allogeneic cell.
[0024] For muscle applications,
e.g., muscle tears, muscle strains, or muscle pulls, the hydrogels (seeded with or without
cells) are injected at the site of injury. Suitable compositions for muscle applications
include a composition comprising a first hydrogel and a second hydrogel, wherein the
first hydrogel degrades at least 10% faster than the second hydrogel, and wherein
the first hydrogel or the second hydrogel comprises a bioactive factor for use in
muscle repair, regeneration, or formation. For example, the bioactive factor comprises
MyoD.
[0025] In some cases, the composition recruits cells to promote muscle or cartilage repair,
regeneration, or formation. Alternatively, the first hydrogel or the second hydrogel
comprises an isolated muscle cell selected from the group consisting of a skeletal
muscle cell, a cardiac muscle cell, a smooth muscle cell, and a myo-progenitor cell.
The isolated muscle cell is an autologous cell or an allogeneic cell.
[0026] For dermal applications,
e.g., burns, abrasions, lacerations, or disease, the hydrogels (seeded with cells or without
cells) are applied directly to the site as poultice or wound dressing. Preferably,
the majority of porogens (
e.g., more than 50%, more than 60%, more than 70%, more than 80%, or more than 90%) within
the bulk hydrogels are directed toward the skin surface and into the skin tissue when
applied directly to the site (
e.g., the burn). In this manner, the bioactive factors or cells are released into the surface
of the skin or lower layers of the skin, and do not migrate away from the skin or
target tissue. An exemplary bioactive factor for use in skin repair, regeneration,
or formation is FGF.
[0027] In some cases, the composition recruits cells to promote skin or cartilage repair,
regeneration, or formation. Alternatively, the first hydrogel or the second hydrogel
comprises an isolated skin cell selected from the group consisting of a fibroblast,
a dermal cell, an epidermal cell, or a dermal progenitor cell. The isolated skin cell
is an autologous cell or an allogeneic cell.
[0028] Bioactive factors such as polynucleotides, polypeptides, or other agents are purified
and/or isolated. Specifically, as used herein, an "isolated" or "purified" nucleic
acid molecule, polynucleotide, polypeptide, or protein, is substantially free of other
cellular material, or culture medium when produced by recombinant techniques, or chemical
precursors or other chemicals when chemically synthesized. Purified compounds are
at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation
is at least 75%, more preferably at least 90%, and most preferably at least 99%, by
weight the compound of interest. For example, a purified compound is one that is at
least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired compound
by weight. Purity is measured by any appropriate standard method, for example, by
column chromatography, thin layer chromatography, or high-performance liquid chromatography
(HPLC) analysis. A purified or isolated polynucleotide (ribonucleic acid (RNA) or
deoxyribonucleic acid (DNA)) is free of the genes or sequences that flank it in its
naturally-occurring state. Purified also defines a degree of sterility that is safe
for administration to a human subject,
e.g., lacking infectious or toxic agents.
[0029] Similarly, by "substantially pure" is meant a nucleotide or polypeptide that has
been separated from the components that naturally accompany it. Typically, the nucleotides
and polypeptides are substantially pure when they are at least 60%, 70%, 80%, 90%,
95%, or even 99%, by weight, free from the proteins and naturally-occurring organic
molecules with they are naturally associated.
[0030] An "isolated nucleic acid" is a nucleic acid, the structure of which is not identical
to that of any naturally occurring nucleic acid, or to that of any fragment of a naturally
occurring genomic nucleic acid spanning more than three separate genes. The term covers,
for example: (a) a DNA which is part of a naturally occurring genomic DNA molecule,
but is not flanked by both of the nucleic acid sequences that flank that part of the
molecule in the genome of the organism in which it naturally occurs; (b) a nucleic
acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote
in a manner, such that the resulting molecule is not identical to any naturally occurring
vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment,
a fragment produced by polymerase chain reaction (PCR), or a restriction fragment;
and (d) a recombinant nucleotide sequence that is part of a hybridgene,
i.e., a gene encoding a fusion protein. Isolated nucleic acid molecules according to the
present invention further include molecules produced synthetically, as well as any
nucleic acids that have been altered chemically and/or that have modified backbones.
[0031] A small molecule is a compound that is less than 2000 daltons in mass. The molecular
mass of the small molecule is preferably less than 1000 daltons, more preferably less
than 600 daltons, e.g., the compound is less than 500 daltons, 400 daltons, 300 daltons,
200 daltons, or 100 daltons.
[0032] The transitional term "comprising," which is synonymous with "including," "containing,"
or "characterized by," is inclusive or open-ended and does not exclude additional,
unrecited elements or method steps. By contrast, the transitional phrase "consisting
of excludes any element, step, or ingredient not specified in the claim. The transitional
phrase "consisting essentially of limits the scope of a claim to the specified materials
or steps "and those that do not materially affect the basic and novel characteristic(s)"
of the claimed invention.
[0033] Other features and advantages of the invention will be apparent from the following
description of the preferred embodiments thereof, and from the claims. Unless otherwise
defined, all technical and scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described herein can be used
in the practice or testing of the present invention, suitable methods and materials
are described below. In the case of conflict, the present specification including
definitions, will control. In addition, the materials, methods, and examples are illustrative
only and not intended to be limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034]
Figure 1 is a series of schematics, photographs, bar graphs, and line graphs showing
the formation of pores in situ within hydrogels as demonstrated via imaging and mechanical
properties testing. Figure 1A is a schematic illustrating the formation of hydrogels.
Left: micro-beads comprised of rapidly degradable hydrogels (red spheres) and mesenchymal
stem cells (MSCs; green). Middle: micro-beads and MSCs are mixed with a second hydrogel
forming polymer material ("bulk gel;" gray), which is crosslinked around the beads.
Right: after degradation of the micro-beads in situ, an intact hydrogel network remains
with a network of pores through which MSCs are released. Figure 1B is a fluorescence
micrograph of fluorescein (green) labeled porogens immediately after fabrication.
Figure 1C is a fluorescence micrograph of fluorescein (green) labeled porogens after
encapsulation into an alginate hydrogel network. Figure 1D is a bar graph illustrating
elastic modulus measurements of either standard hydrogels (blue bars) or pore-forming
hydrogels (red bars). At day 0, there is no statistically significant difference in
the overall rigidity of pore-forming composites and standard hydrogels because pores
have not formed; however, after 4 days, the modulus of the composite drops substantially
because of pore formation. Figure 1E is a series of scanning electron micrographs
depicting pore-forming hydrogels immediately after formation (Day 0) showing a grossly
intact network, 5 days after fabrication, or 10 days after fabrication, at which time
significant pore formation was observed. Figure 1F and Figure 1G verify that the elastic
modulus of the composite material (50% porogen volume fraction) is not substantially
different from the elastic modulus of a standard hydrogel (no porogens), but that
as voids form, the modulus of the composite drops substantially. The decrease in composite
elastic modulus at one week corresponds to the density of voids, and at low porogen
density, there is a linear relationship between the density of voids and decrease
in composite elastic modulus. Figures H and I illustrate that the fracture toughness
of the composite material (25% porogen volume fraction) is initially similar to the
fracture toughness of a standard hydrogel with no porogen, but shortly decreases to
a fraction of the initial value. As with elastic modulus, the decrease in fracture
toughness scales with the density of porogen, though in a non-linear manner. Scale
bars: (B, C, and E): 1 mm.
Figure 2 is a series of photographs, line graphs, and schematics illustrating mesenchymal
stem cell deployment in-vitro. Specifically, Figure 2 demonstrates stem cell release
from pore-forming hydrogels in vitro, which also shows that release can be tuned by varying the composition of porogens
and the compartmentalization of cells within porogens versus bulk. Figure 2A is a
line graph showing the kinetics of interconnected void formation assessed with a capillary
assay (error bars are standard error of the mean, n = 3-4). Interconnected voids formed
over the first 7 days unless a very high fraction of porogens (above the percolation
limit; 80%) were present. No substantial interconnected void formation was observed
with a sub-percolation porogen density. Figure 2B is a series of schematics and photographs
showing 3-dimensional reconstructions of Calcein-AM stained cells distributed throughout
pore-forming hydrogels. The substantial changes in cell morphology depict the cells'
ability to migrate and proliferate within pore-forming hydrogels, whereas cells remained
sparse and rounded within standard hydrogels. Ki-67 immunofluorescence (green) indicates
increased proliferation, while nuclear counterstain (Hoescht, blue) higher cellularity,
within pore-forming hydrogels compared to standard hydrogels. Figure 2C is a set of
fluorescence micrographs showing the effects of void formation on cellularity and
cell morphology within pore forming hydrogels. Specifically, fluorescence micrographs
of pore-forming hydrogels were stained for live mesenchymal stem cells (MSC) (Calcein-AM,
green) or dead cells (Ethidium Homodimer, red) after 4-10 days in vitro. The spherical cell morphology denotes cells confined in a nanoporous material, and
is present at short time frames in both materials, but only in standard hydrogels
over longer time frames. Figure 2D is a line graph showing the cumulative number of
MSC released after 12 days as a function of porogen volume density. Figure 2E is a
line graph and schematic illustrating the kinetics of deployment for MSC encapsulated
either into the bulk phase, the porogen phase of pore-forming scaffolds, or into standard
hydrogels. Porogens were prepared with 7.5% oxidized alginate and crosslinked in 100
mM CaCl2. Figure 2F is a line graph showing the kinetics of MSC deployment from the porogen
phase of pore-forming hydrogels as a function of porogen fabrication conditions for
D1 cells encapsulated into porogens. Figure 2G is a bar graph showing that quantitative
analysis of 3H-thymidine incorporation indicates enhanced cell proliferation, in an RGD-dependent
manner. Figure 2H is a series of line graphs showing the effects of the degree of
alginate oxidation degree on cell release. At a constant level of calcium to crosslink
porogens (100 mM), increasing the degree of oxidation from 3-7.5% substantially increased
the overall number of release cells, whereas lowered the degree of oxidation slightly
delayed cell release. At a constant degree of porogen degree of oxidation (7.5%),
increasing the concentration of calcium used to crosslink porogens from 25-100mM lowered
the overall number of released cells and slightly delayed the onset of cell release.
Scale bars: (A): 100 µm.
Figure 3 is a series of photographs and line graphs showing the results of controlling
mesenchymal stem cell deployment, engraftment and proliferation in vivo. Specifically, Figure 3 demonstrates stem cell release from pore-forming hydrogels
in vivo within the subcutaneous space of nude mice. Figure 3A is a photograph showing representative
images of Nude mice into which 2 x 106 mCherry-expressing MSC were deployed, either 7 or 30 days after injection in standard
hydrogels (left), pore-forming hydrogels in which porogens were crosslinked with either
100 mM CaCl2 (center), or saline (right). The bulk component of hydrogels was modified with 2
RGD / polymer chain. Initially, more cells engrafted in the saline only condition,
but at later time-points, there were fewer cells in this condition and substantially
more cells eventually engrafting when released from pore-forming hydrogels. Figure
3B is a line graph showing the quantification of the relative radiant efficiency (proportional
to cell density) of mCherry-MSC injected in pore-forming hydrogels, standard hydrogels,
or saline. Figure 3C is a line graph showing that decreasing the density of calcium
used to crosslink porogens substantially decreased the overall density of released
cells, and slightly delayed the kinetics of deployment (error bars are SEM, n=4-6).
Figure 3D is a series of photographs showing the ability of pore-forming hydrogels
to enhance human mesenchymal stem cell mediated bone regeneration using a Nude Rat
cranial defect model. Critically-sized defects were formed in the crania of Nude Rats
(Charles River). Immediately after defect formation, commercially available human
mesenchymal stem cells (Lonza) were transplanted into the defect space, either within
saline ("Cells Only"), a standard hydrogel (2 RGD / alginate polymer, 60 kPa), or
a Pore-Forming Hydrogel. Representative cross-sections of micro-computed tomographic
analysis of new bone formation within cranial defects 4 weeks after implantation.
Substantially more new bone forms within defects in which cells were delivered via
pore-forming hydrogels.
Figure 4 is a series of photomicrographs depicting the use of pore-forming hydrogels
to release distinct populations at different times. Figure 4A-Figure 4C are fluorescent
micrographs of GFP-expressing myoblasts and outgrowth endothelial cells (OECs) adherent
to tissue culture plastic after 4 days of culture within pore-forming hydrogels in
which the chemistry used to form porogens was varied and the different cell types
were initially placed into distinct compartments: (Figure 4A): myoblasts in bulk component,
OECs in porogen component; (Figure 4B): myoblasts in bead component, OECs in porogen
component; (Figure 4C): both myoblasts and OECs in bulk component. Figure 4D is a
representative micrograph of a plastic substrate on which equal numbers of GFP-myoblasts
and OECs were seeded. Myoblasts outgrew OECs. Cells were stained with Ethidium Homodimer
(red), so that GFP-myoblasts appear yellow and OECs appear red. Images taken at 10x
magnification.
Figure 5 is a series of photomicrographs depicting using pore-forming hydrogels for
chemokine-mediated cell recruitment. Specifically, Figure 5 shows the controlling
chemokine-mediated cell recruitment by pore-forming hydrogels in vivo. Alginate was first oxidized and then reduced with sodium borohydride to make alcohol
groups that replace what were originally sugars. Figure 5A and Figure 5B are fluorescent
micrographs of dendritic cell recruitment into (Figure 5A) standard, injectable alginate
gel, and (Figure 5B) pore-forming hydrogel. Both sets of hydrogels were loaded with
2 pg of granulocyte-macrophage colony stimulating factor. Figure 5C and 5D are fluorescent
micrographs of dendritic cell recruitment into pore-forming hydrogels fabricated with
(Figure 5C) oxidized or (Figure 5D) reduced porogens. No chemokine was added. For
histology, dendritic cells are stained for CDllc (green) and NIHC-II (red), with Hoescht
nuclear counterstain (blue). In Figure 5C and Figure 5D, only nuclear staining (white)
was performed. This figure shows the difference in host cell recruitment by materials
with porogens formed from the oxidized alginate vs. reduced alginate.Scale bars: 100
pm.
Figure 6 is a series of line graphs, bar charts, and photographs demonstrating the
control of stem cell proliferation within pore-forming hydrogels, deployment from
hydrogels, and ability to regenerate bone by varying bulk phase composition. Figures
6A and Figure 6B are line graphs showing the analysis of 24 hr 3H-thymidine incorporation
(proportional to DNA synthesis) by mesenchymal stem cells (D1; red curve) or cumulative
MSC deployment (blue curves) from pore-forming hydrogels after 7 days of culture as
a function of (Figure 6A) density of RGD peptides in bulk gels with 60 kPa modulus,
or (Figure 6B) elastic modulus of bulk hydrogels presenting 10 RGD peptides / alginate
polymer (data are mean +/- SEM, n = 3-5). RGD density had significant effects of cell
proliferation, whereas elastic modulus had effects on both proliferation and release
(p < 0.05, ANOVA). Figure 6C is a bar graph showing the analysis of DNA synthesis
as a function of pore formation. Figure 6D is a series of photographs showing staining
for Ki-67 (proliferation marker, green) in D1 cells in cryosectioned pore-forming
hydrogels after 50 days of culture. Figure 6E and 6F is a line graph showing the control
of mesenchymal stem cell deployment, engraftment and proliferation in vivo. Figure 6E is a line graph showing the quantification of the relative radiant efficiency
(proportional to cell density) of mCherry-MSC injected in pore-forming hydrogels in
which the bulk phase was modified with either 2 (
), or 10 (
) RGD peptides per alginate polymer. Alternatively, cells were injected in a standard
hydrogel with 2 RGD peptides / alginate polymer (▲). Figure 6F shows the quantification
of the percentage of healing (new bone formation due to human MSC transplanted into
Nude Rat cranial defects) as a function of the method of MSC delivery. Error bars
are SEM, n = 4-6.
Figure 7 is a series of line graphs and bar frequency charts showing the mechanical
properties and in-vitro degradation of hydrogels formed from binary alginates. Figures 7A and Figure 7B show
elastic moduli (Figure 7A) and degradation (Figure 7B) of bulk hydrogels formed by
crosslinking binary combinations of oxidized alginate (5% theoretical degree of oxidation)
at a constant density of 20 mg/mL with unmodified, high Mw alginate. Degradation was assessed by comparing the dry mass after 4 days in-vitro to initial dry mass. Figure 7C is a histogram of diameters of porogens formed from
binary mixtures of 20 mg/mL oxidized alginate with 7.5 mg/mL unmodified alginate.
Porogen diameter was measured by processing fluorescent micrographs of porogens prepared
from aminofluorescenin-labeled alginates. Error bars are SD, n = 3-4.
Figure 8 is a schematic of an implantable biomaterial that mimics certain aspects
of stem-cell niches in that it activates transplanted progenitor cells to proliferate
and programs them to differentiate into cells that migrate into damaged tissues to
participate in regeneration.
Figure 9 is a schematic of a hydrogel scaffold (left, top), which controls transplanted
cell fate through presentation of specific cues, but prevents transplanted cells from
migrating out of, and host cells from migrating into, the material. Bottom: example
data depicting the ability of a nanoporous hydrogel to control mesenchymal stem cell
fate, in this case via elastic modulus. Right, top: schematic of a macroporous sponge
which controls transplanted fate through presentation of specific cues, while also
allowing host cells to migrate into, and transplanted cells to migrate out of the
material.
Figure 10 is a series of images depicting an alternative strategy to produce macroporous
hydrogels. As described in the schematics (center, right), porogens are embedded into
a "bulk" hydrogel, or photolithographic techniques are applied, to provide non-crosslinked
regions of the bulk hydrogel. After crosslinking the bulk hydrogel, the non-crosslinked
portions of the hydrogel and porogens are removed using solvents such as acetone.
Left: image of a macroporous hydrogel.
Figure 11 is a schematic and a bar chart depicting a strategy to create rapidly degrading
alginate-based hydrogel porogens. Top left: chemical reaction scheme to oxidize alginate
to alginate dialdehyde with NaIO4. Top right: schematic depicting the loss of crosslinkable, guluronic-acid rich portions
of alginate (short, straight segments), and the overall decrease in polymer Mw due to sodium periodate oxidation. Bottom: data depicting loss in dry mass over time
from hydrogels made from 20mg/mL non-modified alginate (squares), 20mg/mL alginate
dialdehyde (5% degree of oxidation; diamonds) or a binary mixture of 20mg/mL alginate
dialdehyde with 7.5mg/mL unmodified alginate (triangles).
Figure 12 is a schematic illustrating porogen fabrication and characterization.
Figure 13 is a schematic showing the control of host cell recruitment with pore forming
hydrogels. Specifically, shown in this Figure is a schematic of an implantable biomaterial
system that mimics the icroenvironment of an infection, allowing the recruitment,
programming and subsequent targeting of activated antigen-presenting dendritic cells
to the lymph nodes to participate in a potent antitumour response.
Figure 14 is a series of photomicrographs showing how the polymers used to comprise
the porogen phase affect the degradation and processing of porogens. Specifically,
shown in this Figure are fluorescence micrographs of porogens formed using fluorescein-labeled
alginate dialdehyde. Immediately after crosslinking in 100mM CaCl2 (top), porogens are grossly intact, whether made using 20mg/mL alginate dialdehyde
(top left) or a binary mixture of 20mg/mL alginate dialdehyde with 7.5mg/mL unmodified
alginate. However, after processing steps used to purify porogens and remove excess
CaCl2, porogens made with purely alginate dialdehyde were damaged, resulting in substantial
change in morphology and release of fluorescein-labeled polymers into solution to
yield a substantial level of background fluorescein fluorescence (bottom left). In
contrast, binary mixtures of 20mg/mL alginate dialdehyde with 7.5mg/mL unmodified
alginate resulted in porogens that could withstand processing steps.
DETAILED DESCRIPTION OF THE INVENTION
[0035] Over the recent decades, biocompatible polymers have been used to form scaffolds
that act as carriers for cell transplantation, or to recruit host cell populations
into the device. Generally, sponges such as poly(lactide-co-glycolide) (PLGA), or
synthetic hydrogels such as alginate are used. However, both sets of materials have
disadvantages. For example, sponges typically adsorb serum proteins, so it is difficult
to control presentation of adhesive proteins or peptides (for example, RGD) from the
material. Sponge materials also typically are not amenable to injection, and require
an invasive surgery for implantation, and also expose transplanted or host cells to
a host environment that may initially be hostile (for example, neutrophils present
during inflammation may attack stem cells). On the other hand, synthetic hydrogels
are typically injectable, allowing minimally-invasive delivery, and do not interact
with proteins. However, prior to the invention described herein, the pore size in
hydrogels was typically much smaller than the diameter of a eukaryote cell, making
it difficult to expand a transplanted cell population, release transplanted cells
to allow them to repair damaged tissues, or recruit host cells into the device.
[0036] Described herein is a method to form pores in situ within hydrogels following hydrogel
injection. Pores form in situ via degradation of sacrificial porogens encapsulated
within the surrounding hydrogel. The kinetics and onset of pore formation are controlled
by manipulating material used to form porogens, and cells are encapsulated either
into the porogens themselves or the hydrogel surrounding them. Examples demonstrate
in vitro deployment, proliferation, and differentiation of stem cells, as well as
in vivo stem cell deployment and chemokine-mediated cell recruitment. The system mediates
controlled deployment of cells out of, or local recruitment of cells into, a polymer
matrix via formation of pores within this matrix. The size, distribution, and formation
kinetics of the pores are predetermined by the user, while the integrity of the matrix
surrounding pores, along with cells or biological factors inside this matrix, are
unchanged.
[0037] Accordingly, described herein is the use of insoluble cues such as hydrogel adhesion
ligand presentation and/or elastic modulus {i.e., stiffness) to generate materials
which are 1) injectable; 2) allow the user to control cell fate using insoluble cues;
and 3) form pores over time to deploy or recruit cells. Specifically, the methods
described herein create pore-forming hydrogels, using a process that allows cells
to be encapsulated into either the pore-forming phase (hereafter referred to as "porogen")
or the non- or slowly-degrading phase (hereafter referred to as "bulk").
[0038] Described herein are methods for a generalized approach to create pore-forming hydrogels
that allow cell encapsulation, and a means to control the kinetics of cell deployment
out of, or recruitment into, the hydrogel. Hydrogel micro-bead "porogens" are formed,
and are next encapsulated into a second, "bulk" hydrogel. The composition of polymers
used to form porogen and bulk hydrogels may be varied; however, the porogen must degrade
more rapidly {e.g., 10%, 20%, 50%, 2X, 5X, 10X, 20X or faster) than the bulk hydrogel.
Cells or bioactive factors (
e.g., growth factors such as granulocyte/macrophage colony stimulating factor (GM-CSF),
vascular endothelial growth factor (VEGF), condensed oligonucleotides,
e.g., CpG, or plasmid DNA ) are optionally encapsulated either into the porogen phase,
bulk hydrogel phase, or into both phases. The porogens degrade in situ over a time-course
pre-determined by the user, at which point cells are released, or may migrate into
the material. However, because they initially lack pores, pore-forming hydrogels are
useful to provide mechanical support immediately after formation (Figure 1).
[0039] Cellular release or recruitment is manipulated by controlling the kinetics of porogen
degradation. For example, the alginate polymers are oxidized to produce alginate dialdehyde,
and the total number of cells released increases as the extent of oxidation increases
(Figure 2, Figure 3). Alternatively, conditions used to crosslink the porogens are
altered to manipulate the time at which significant porogen degradation and cell release
begin to occur (Figure 2). Porogen chemistry can further be varied to facilitate,
or inhibit, interaction with host proteins (Figure 5).
[0040] Cell release and cell fate are controlled by manipulating the biophysical and biochemical
properties (
e.g. elastic modulus and density of integrin-binding adhesion peptides such as RGD) of
the bulk hydrogel. For example, pore formation, bulk hydrogel RGD density and bulk
hydrogel elasticity all affect cell proliferation within these materials (Figure 2,
Figure 6). The orthogonal processing of porogens and bulk separate from one another
enhances the ability to tune the system to manipulate cell release and cell fate.
For example, stem cell lineage commitment is modulated by varying elastic modulus
or RGD density, independent of the kinetics of pore formation. In contrast, other
techniques used to form macro-porous materials (
e.g. solvent-based extraction of porogens) are not compatible with cell encapsulation,
and typically affect the physical properties of both the porogen and bulk phases.
The physical and biochemical properties rapidly degrading bulk hydrogel materials
change continuously over the course of degradation. These parameters are harnessed
to design the bulk phase to regulate cell fate.
Hydrogel compositions
[0041] Hydrogels comprise a network of polymer chains that are hydrophilic. Hydrogel (also
called aquagel) is sometimes found as a colloidal gel in which water is the dispersion
medium. Hydrogels are highly absorbent (they can contain over 99.9% water) natural
or synthetic polymers. Hydrogels also possess a degree of flexibility very similar
to natural tissue, due to their significant water content. Hydrogel is comprised of
cross-linked polymers. Exemplary hydrogels are comprised materials that are compatible
with cell encapsulation such as alginate, polyethylene glycol (PEG), PEG-acrylate,
agarose, and synthetic protein (
e.g., collagen or engineered proteins (
i.e., self-assembly peptide-based hydrogels). For example, a commercially available hydrogel
includes BD™ PuraMatrix™ Peptide Hydrogel, which is a synthetic matrix that is used
to create defined three dimensional (3D) micro-environments for cell culture.
[0042] For example, the hydrogel is a biocompatible polymer matrix that is biodegradable
in whole or in part. Examples of materials which can form hydrogels include alginates
and alginate derivatives, polylactic acid, polyglycolic acid, poly(lactic-co-glycolic
acid) (PLGA) polymers, gelatin, collagen, agarose, natural and synthetic polysaccharides,
polyamino acids such as polypeptides particularly poly(lysine), polyesters such as
polyhydroxybutyrate and poly-epsilon.-caprolactone, polyanhydrides; polyphosphazines,
poly(vinyl alcohols), poly(alkylene oxides) particularly poly(ethylene oxides), poly(allylamines)(PAM),
poly(acrylates), modified styrene polymers such as poly(4-aminomethylstyrene), pluronic
polyols, polyoxamers, poly(uronic acids), poly(vinylpyrrolidone), and copolymers of
the above, including graft copolymers. Synthetic polymers and naturally-occurring
polymers such as, but not limited to, collagen, fibrin, hyaluronic acid, agarose,
and laminin-rich gels may also be used.
[0043] A preferred material for the hydrogel is alginate or modified alginate material.
Alginate molecules are comprised of (1-4)-linked β-D-mannuronic acid (M units) and
α L-guluronic acid (G units) monomers, which can vary in proportion and sequential
distribution along the polymer chain. Alginate polysaccharides are polyelectrolyte
systems which have a strong affinity for divalent cations (
e.g., Ca
+2, Mg
+2, Ba
+2) and form stable hydrogels when exposed to these molecules.
[0044] Synthetic hydrogels are typically injectable, allow for minimally-invasive delivery,
and do not interact with proteins. Hence, the presentation of adhesion proteins or
peptides is precisely controlled. Moreover, synthetic hydrogels typically have a pore
mesh size that is much smaller than cells (< 10 nm, whereas cells are > 10um), which
prevents host cells from attacking transplanted cells. However, this small pore size
also prevents transplanted cells from proliferating extensively within the material,
and also precludes their eventually being released to affect various functions (for
example, regeneration of functional tissue or destruction of diseased tissue).
[0045] Several techniques have been introduced to combine desirable features of hydrogels
and sponges - for example, rigid microspheres have been encapsulated into hydrogels,
and then extracted with solvents (
e.g. acetone) to leave behind a macroporous hydrogel, and freeze-drying has been applied
to generate macroporous hydrogels. Hydrogels can be modified to rapidly degrade
in vivo to release host cells. However, prior to the invention described herein, none of
the approaches allowed for the combination of a non-degrading (or slowly degrading)
material component with cell encapsulation. The mechanical properties and biochemical
composition of hydrogel materials strongly affect cell fate, and degradation in-and-of
itself may intrinsically regulate cell fate.
Pore-forming compositions
[0046] Hydrogel micro-beads ("porogens") are formed. Next, porogens are encapsulated into
a "bulk" hydrogel that is either non-degradable or which degrades at a slow rate compared
to the porogens. Cells are optionally encapsulated either into the porogen or bulk
compartment. Immediately after hydrogel formation, or injection into the desired site
in vivo, the composite material lacks pores, and serves as a surgical bulking agent. Subsequently,
porogen degradation causes pores to form in situ, and encapsulated cells deploy away
from the composite material and into surrounding tissues or remote tissues,
e.g., lymph nodes, in the body. The size and distribution of pores are controlled during
porogen formation, and mixing with the polymers which form the bulk hydrogel.
[0047] Alternatively, the hydrogel is injected without encapsulated cells, and pore formation
is used as a means of recruiting host cells, in combination or independent of chemokines
released from either the bulk or porogen component. The porogens are comprised of
any biocompatible polymer, as long as they degrade more rapidly than the material
used to form the "bulk" hydrogel, and are initially mechanically stable enough to
withstand being mixed with the polymer which forms the bulk hydrogel phase. The "bulk"
is comprised of any hydrogel-forming polymer.
Alginate compositions
[0048] The polymers utilized in the compositions and methods are naturally-occurring or
synthetically made. In one example, both the porogens and bulk hydrogels are formed
from alginate. "Alginate" as that term is used here, refers to any number of derivatives
of alginic acid (e.g., calcium, sodium or potassium salts, or propylene glycol alginate).
See,
e.g., PCT/US97/16890.
[0049] The alginate polymers suitable for porogen formation have a Dalton molecular weight
from 5,000 to 500,000 Da. The polymers are optionally further modified (e.g., by oxidation
with sodium periodate, (
Bouhadir et al., 2001, Biotech. Prog. 17:945-950), to facilitate rapid degradation. In the examples described below, the polymers
were crosslinked by extrusion through a nebulizer with co-axial airflow into a bath
of divalent cation (for example, Ca2+ or Ba2+) to form hydrogel micro-beads. The higher
the airflow rate, the lower the porogen diameter.
[0050] The concentration of divalent ions used to form porogens may vary from 5 to 500 mM,
and the concentration of polymer from 1% to 5% by weight. However, any method which
produces porogens that are significantly smaller than the bulk phase is suitable.
Porogen chemistry can further be manipulated to produce porogens that have a some
interaction with host proteins and cells (
e.g., alginates oxidized to an extent of >5% of sugar resides interact significantly with
host cells, Figure 5), or to inhibit this interaction (
e.g., oxidized alginates that are reduced with NaBH4 exhibit minimal interaction with protein
or with host cells, Figure 5).
[0051] The alginate polymers suitable for formation of the bulk hydrogel have a Dalton molecular
weight from 5,000 to 500,000 Da. The polymers may be further modified (for example,
by oxidation with sodium periodate), to facilitate degradation, as long as the bulk
hydrogel degrades more slowly than the porogen. The polymers may also be modified
to present biological cues to control cell responses (
e.g., integrin binding adhesion peptides such as RGD). Either the porogens or the bulk
hydrogel may also encapsulate bioactive factors such as oligonucleotides, growth factors
or drugs to further control cell responses. The concentration of divalent ions used
to form the bulk hydrogel may vary from 5 to 500 mM, and the concentration of polymer
from 1% to 5% by weight. The elastic modulus of the bulk polymer is tailored,
e.g., to control the fate of encapsulated cells.
Example 1: Forming Pores in situ within Hydrogels
[0052] The formation of pores in situ within hydrogels as demonstrated via imaging and mechanical
properties testing is shown in Figure 1. As shown in Figure 1A, micro-beads comprised
of rapidly degradable hydrogels (red spheres) were mixed with a second hydrogel forming
polymer material, which is crosslinked around the beads. After degradation of the
micro-beads in situ, an intact hydrogel network (pink) remained with a network of
pores. The elastic modulus measurements of either standard hydrogels (left bars) or
pore-forming hydrogels (right bars) were determined (Figure ID). At day 0, there was
no statistically significant difference in the overall rigidity of pore-forming composites
and standard hydrogels because pores have not formed; however, after 4 days, the modulus
of the composite drops substantially because of pore formation.
[0053] Additional methods relevant to generating the hydrogels described herein are as follows.
Bouhadir et al. Polymer 1999; 40: 3575-84 describes the oxidation of alginate with sodium periodate, and characterizes the
reaction.
Bouhadir et al. Biotechnol. Prog. 2001; 17: 945-50 describes oxidation of high molecular weight alginate to form alginate dialdehyde
(alginate dialdehyde is high Mw alginate in which a certain percent, {e.g., 5%), of
sugars in alginate are oxidized to form aldehydes), and application to make hydrogels
degrade rapidly.
Kong et al. Polymer 2002; 43: 6239-46 describes the use of gamma-irradiation to reduce the weight-averaged molecular weight
(M
w) of guluronic acid (GA) rich alginates without substantially reducing GA content
{e.g., the gamma irradiation selectively attacks mannuronic acid, MA blocks of alginate).
Alginate is comprised of GA blocks and MA blocks, and it is the GA blocks that give
alginate its rigidity (elastic modulus).
Kong et al. Polymer 2002; 43: 6239- 46 shows that binary combinations of high M
w, GA rich alginate with irradiated, low M
w, high GA alginate crosslinks with calcium to form rigid hydrogels, but which degrade
more rapidly and also have lower solution viscosity than hydrogels made from the same
overall weight concentration of only high M
w, GA rich alginate.
Alsberg et al. J Dent Res 2003; 82(11): 903-8 describes degradation profiles of hydrogels made from irradiated, low M
w, GA-rich alginate, with application in bone tissue engineering.
Kong et al. Adv. Mater 2004; 16(21): 1917-21 describes control of hydrogel degradation profile by combining gamma irradiation
procedure with oxidation reaction, and application to cartilage engineering.
[0054] Techniques to control degradation of hydrogen biomaterials are well known in the
art. For example,
Lutolf MP et al. Nat Biotechnol. 2003; 21: 513-8 describes poly(ethylene glycol) based materials engineered to degrade via mammalian
enzymes (MMPs).
Bryant SJ et al. Biomaterials 2007; 28(19): 2978-86 (
US 7,192,693 B2) describes a method to produce hydrogels with macro-scale pores. A pore template
{
e.g., poly-methylmethacrylate beads) is encapsulated within a bulk hydrogel, and then acetone
and methanol are used to extract the porogen while leaving the bulk hydrogel intact.
Silva et al. Proc. Natl. Acad. Sci USA 2008; 105(38): 14347-52;
US 2008/0044900 describes deployment of endothelial progenitor cells from alginate sponges. The sponges
are made by forming alginate hydrogels and then freeze- drying them (ice crystals
form the pores). These materials improve the therapeutic effect of the cells (compared
to cells delivered alone), but these materials must be implanted surgically
(i.e., non-injectable), are not amenable to cell encapsulation (cells will die when freeze
dried), and this strategy makes it difficult to control cell fate by controlling elastic
modulus.
Ali et al. Nat Mater 2009 describes the use of porous scaffolds to recruit dendritic cells and program them
to elicit anti-tumor responses.
Huebsch et al. Nat Mater 2010; 9: 518-26 describes the use of hydrogel elastic modulus to control the differentiation of encapsulated
mesenchymal stem cells.
[0055] Described herein is the use of insoluble cues such as hydrogel adhesion ligand presentation
and/or elastic modulus
(i.e., stiffness) to generate materials which are 1) injectable; 2) allow the user to control
cell fate using insoluble cues; and 3) form pores over time to deploy or recruit cells.
Specifically, the methods described herein create pore-forming hydrogels, using a
process that allows cells to be encapsulated into either the pore-forming phase (hereafter
referred to as "porogen") or the non- or slowly-degrading phase (hereafter referred
to as "bulk"). In the methods described herein, the porogen degrades by hydrolysis
rather than by solvents, which means that cells are encapsulated either into the porogen
or the bulk gel around them, and there is very little chance that proteins or other
bioactive compounds encapsulated into the gel would be denatured.
[0056] As described in detail below, porogens stayed intact during encapsulation, but rapidly
degraded to yield voids that were visible by scanning electron microscopy, and resulted
in loss of elastic modulus and fracture toughness of the composite materials. Specifically,
scanning electron micrographs (SEM) showed that pore-forming hydrogels immediately
after formation (Day 0) possessed a grossly intact network; however, by 10 days after
fabrication, significant pore formation was observed (Figure IE). The elastic modulus
of the composite material (50% porogen volume fraction) was not substantially different
from the elastic modulus of a standard hydrogel (no porogens); however, as voids form,
the modulus of the composite drops substantially (Figure 1F and Figure 1G). The decrease
in composite elastic modulus at one week corresponds to the density of voids, and
at low porogen density, there is a linear relationship between the density of voids
and decrease in composite elastic modulus. The fracture toughness of the composite
material (25% porogen volume fraction) was initially similar to the fracture toughness
of a standard hydrogel with no porogen, but it decreases to a fraction of the initial
value (Figures H and I). As with elastic modulus, the decrease in fracture toughness
scales with the density of porogen, though in a non-linear manner. These results demonstrate
that porogens stay intact during encapsulation and degrade in situ to form voids.
Example 2: In vitro and in vivo release of cells
[0057] Pore-forming hydrogels were formed by encapsulating degradable alginate porogens,
along with bone marrow stromal stem cells (D1) into high molecular weight bulk alginate
gel. Porogens were formed with a binary mixture of 20mg/mL of alginate dialdehyde
(theoretical oxidation of 7.5% of alginate sugar residues in high Mw, high guluronic
acid content alginate) and 7.5mg/mL high Mw, high guluronic acid (GA) content alginate.
This polymer mixture was extruded through a glass nebulizer with co-axial nitrogen
airflow into a bath of 0.1M CaCl
2 and 0.1M HEPES to crosslink polymers. Porogens were washed extensively with serum
free cell culture medium. The bulk hydrogel was formed by 20mg/mL high Mw, high GA-content
alginate modified with 2 RGD peptides per alginate polymer. D1 cells and porogens
were mixed into the bulk hydrogel material using syringes and then the composite was
crosslinked with Calcium Sulfate. The number of D1 cells released from this system
over time
in vitro is shown in Figure 2. The kinetics of release could be modified by 1) controlling
the concentration of CaCl
2 used to form porogens, by 2) varying the composition (degree of oxidation) of porogens,
and by 3) varying the compartmentalization of cells (either within porogens or within
bulk gel).
[0058] Specifically, mesenchymal stem cell deployment in-vitro is illustrated in Figure
2. Stem cells were released from pore-forming hydrogels
in vitro, and this release can be tuned by varying the composition of porogens and the compartmentalization
of cells within porogens versus bulk. The effects of porogen density (0-80 volume
percent) on cellularity and efflux from pore-forming hydrogels are shown in Figure
2C. Specifically, fluorescence micrographs of pore-forming hydrogels were stained
for live mesenchymal stem cells (MSC) (Calcein-AM, green) or dead cells (Ethidium
Homodimer, red) after 4-10 days
in vitro. The spherical cell morphology denotes cells confined in a nanoporous material, and
is present at short time frames in both materials, but only in standard hydrogels
over longer time frames. The cumulative number of MSC released after 12 days as a
function of porogen volume density is shown in Figure 2D.
[0059] As shown in Figure 2D, the size of the porogen is related to the size of the overall
composite material. Specifically, for the material to stay intact, the porogen diameter
is < 10% of the smallest dimension of the overall composite. The density of porogens
is between 10-80 percent of the overall volume for both cell recruitment and cell
release, e.g., between 15% and 75%, between 20% and 70%, between 25% and 65%, between
30% and 60%, or between 35% and 55% of the overall volume. Preferably, the density
of porogens is at least 50% of the overall volume.
[0060] Physical and
in vitro studies were performed to determine the kinetics of interconnected void formation,
and the corresponding kinetics of mesenchymal stem cell release (Figure 2A and Figure
2E, respectively. A clonally derived, commercially available mouse mesenchymal stem
cell line (D1) was used for these
in vitro studies. A capillary assay was used to assess void formation. Briefly, the density
of interconnected voids was measured by first weighing buffer-saturated composite
pore-forming gels, and then re-weighing gels after wicking away water by gently touching
the surface of the gel with a paper towel. The void fraction was calculated based
on the relative change in mass. For
in vitro cell release assays, the bulk component of pore-forming hydrogels was modified with
2 RGD peptides / alginate polymer, and had an elastic modulus of 60 kPa. Interconnected
voids formed over the first 7 days unless a very high fraction of porogens (above
the percolation limit; 80%) were present. No substantial interconnected void formation
was observed with a sub-percolation porogen density. The kinetics of MSC deployment
from the porogen phase of pore-forming hydrogels as a function of porogen fabrication
conditions for D1 cells encapsulated into porogens is illustrated in Figure 2F.
[0061] Release studies were subsequently performed with a mouse MSC line. Cell release was
observed in proportion to overall pore density and gradual change in cell morphology,
reflecting a loss of micron-scale confinement. Experiments were performed to determine
the effects of pore formation on cellularity and cell proliferation within hydrogels.
Cellularity was determined qualitatively using Calcein-AM staining, while proliferation
was determined qualitatively by immunostaining for Ki-67 expression or quantitatively
by measuring
3H-thymidine incorporation. Three-dimensional reconstructions of Calcein-AM stained
cells distributed throughout pore-forming hydrogels are presented in Figure 2B. The
substantial changes in cell morphology depict the cells' ability to migrate and proliferate
within pore-forming hydrogels, whereas cells remained sparse and rounded within standard
hydrogels. Ki-67 immunofluorescence indicated higher cellularity, and increased proliferation,
within pore-forming hydrogels compared to standard hydrogels. Quantitative analysis
of
3H-thymidine incorporation indicated enhanced cell proliferation in an RGD-dependent
manner (Figure 2G).
[0063] Pore-forming hydrogels were formed with a constant bulk component (2 RGD / polymer,
60 kPa), and constant porogen density (50%), but varying porogen composition. The
chemical composition of porogens was manipulated by varying the theoretical degree
of oxidation of the alginate polymers. Oxidation degree was controlled by varying
the ratio of sodium periodate to alginate during the oxidation reaction (Bouhadir
2001). Binary mixtures of 20mg/mL oxidized alginate with 5mg/mL unmodified, high M
w alginate, were used to form porogens. Porogens were formed by crosslinking in a bath
of 25-100mM CaCl
2. The effects of the degree of alginate oxidation degree on cell release are shown
in Figure 2H. At a constant level of calcium to crosslink porogens (100 mM), increasing
the degree of oxidation from 3-7.5% substantially increased the overall number of
release cells, whereas lowering the degree of oxidation slightly delayed cell release.
At a constant degree of porogen degree of oxidation (7.5%), increasing the concentration
of calcium used to crosslink porogens from 25-100 mM lowered the overall number of
released cells and slightly delayed the onset of cell release.
Example 3: Controlling mesenchymal stem cell deployment, engraftment, and proliferation
in vivo
[0064] Finally,
in vivo studies were performed to determine if pore-forming hydrogels could be used to manipulate
the release kinetics of MSC
in vivo. For this, mouse MSC expressing mCherry were transplanted subcutaneously into Nude
mice. Cell engraftment, proliferation and deployment were observed with non-invasive
fluorescence imaging. This showed that not only did pore-forming gels delay engraftment
compared to cells delivered in saline, but that these materials ultimately led to
more proliferation. The hydrogels provide a micro-environment ammenable to proliferation
after pores have formed. Finally, as these materials were useful to promote MSC release
and expansion
in vivo, human MSC were administered to regenerate cranial defects on nude rats. This led
to improved regeneration of mineralized bone, even at an early time-point.
[0065] Specifically, for
in vivo studies, D1 cells were modified to constitutively express a detectable marker,
e.g., mCherry or green fluorescent protein (GFP), and were encapsulated either into standard
bulk gels with no porogens, pore-forming hydrogels, or mixed with saline. Next, cells
were injected into the backs of Nude mice through 18-gauge needles. Cell release and
proliferation over time were monitored via mCherry fluorescence observed on an IVIS
system (Caliper Life Sciences). These data revealed significantly more cell release
and proliferation from pore-forming hydrogels than from standard gels (Figure 3).
Moreover, although cells did proliferate if delivered by simple saline injection,
deployment from pore-forming hydrogels 1) altered the kinetics of local cell delivery
and proliferation, and 2) eventually led to a substantially higher number of delivered
cells (Figure 3).
[0066] Specifically, experiments were performed to determine the ability to manipulate the
kinetics of cell release
in vivo by varying the composition of porogens. As shown in Figure 3, stem cells were released
from pore-forming hydrogels
in vivo within the subcutaneous space of nude mice. 2 x 10
6 mCherry-expressing MSC were deployed into Nude mice, either 7 or 30 days after injection
in standard hydrogels (left), pore-forming hydrogels in which porogens were crosslinked
with either 100 mM or 50 MM CaCl
2 (center), or saline (right). The bulk component of hydrogels was modified with 2
RGD / polymer chain. Initially, more cells engrafted in the saline only condition,
but at later time-points, there were fewer cells in this condition and substantially
more cells eventually engrafting when released from pore-forming hydrogels. The quantification
of the relative radiant efficiency (proportional to cell density) of mCherry-MSC injected
in pore-forming hydrogels, standard hydrogels, or saline is shown in Figure 3B. Decreasing
the density of calcium used to crosslink porogens substantially decreased the overall
density of released cells, and slightly delayed the kinetics of deployment (Figure
3C; error bars are SEM, n=4-6). The ability of pore-forming hydrogels to enhance human
mesenchymal stem cell mediated bone regeneration was demonstrated in a Nude Rat cranial
defect model (Figure 3D). Critically-sized defects were formed in the crania of Nude
Rats (Charles River). Immediately after defect formation, commercially available human
mesenchymal stem cells (Lonza) were transplanted into the defect space, either within
saline ("Cells Only"), a standard hydrogel (2 RGD / alginate polymer, 60 kPa), or
a Pore-Forming Hydrogel. The top row of Figure 3D shows representative cross-sections
of micro-computed tomographic analysis of new bone formation within cranial defects
4 weeks after implantation. Substantially more new bone forms within defects in which
cells were delivered via pore-forming hydrogels. Doxycycline incorporation (green)
into newly-forming bone at 12 weeks after implantation demonstrates that pore-forming
hydrogels lead to positive doxycycline staining within the tissue rather than false-positive
staining of the subcutaneous tissues.
Example 4: In vitro release of two different cell populations at distinct times
[0067] Pore-forming hydrogels were formed as described in Examples 1 and 2. Equal numbers
(approximately 106 cells/mL of composite pore-forming hydrogel) GFP-expressing myoblasts
and outgrowth endothelial cells (OECs, vascular progenitor cells) were encapsulated
into different compartments of the material. After 5 days of culture
in vitro, cells that were released and adherent to tissue culture plastic were stained with
Ethidium Homodimer (EtD-1; red). As shown in Figure 4, the cell type encapsulated
into the bulk gel deployed more rapidly. This pattern of deployment occurred even
for OECs, which migrate more slowly and proliferate less extensively than GFP-myoblasts
(based on analysis of substrates onto which equal numbers of both cell types were
added; Figure 4d).
[0068] Specifically, the use of pore-forming hydrogels to release distinct populations at
different times is shown in Figure 4. Fluorescent micrographs of GFP-expressing myoblasts
and outgrowth endothelial cells (OECs) adherent to tissue culture plastic after 4
days of culture within pore-forming hydrogels in which the chemistry used to form
porogens was varied and the different cell types were initially placed into distinct
compartments are shown in Figures 4A-4C. Figure 4A depicts myoblasts in bulk component,
OECs in porogen component, while Figure 4B depicts myoblasts in bead component, OECs
in porogen component. Figure 4C depicts both myoblasts and OECs in bulk component.
Equal numbers of GFP-myoblasts and OECs were seeded onto a plastic substrate (Figure
4D). Myoblasts outgrew OECs. Cells were stained with Ethidium Homodimer (red), so
that GFP-myoblasts appear yellow and OECs appear red.
Example 5: Recruitment of host lymphocytes from subcutaneous tissues by pore-forming hydrogels with different porogen formulations
[0069] Pore-forming hydrogels were formed as described in Examples 1 and 2. To form the
porogen phase, 7.5mg/mL of high Mw, GA-rich alginate polymer was combined with 20mg/mL
of either alginate dialdehyde (7.5% theoretical degree of oxidation) or alginate dialdehyde
in which aldehyde groups were reduced to alcohol groups. Pore-forming hydrogels without
encapsulated cells were next injected into the backs of C57/BL6 or Balb/c mice. After
14 days, recruitment of host dendritic cells was observed by histology.
[0070] As described in detail below, pore-forming hydrogels were utilized for chemokine-mediated
cell recruitment. Alginate was first oxidized and then reduced with sodium borohydride
to make alcohol groups that replace what were originally sugars. Figure 5A and Figure
5B show a comparison of dendritic cell (DC) recruitment by a standard, degradable
alginate hydrogel versus a pore-forming alginate hydrogel. Both sets of hydrogels
were loaded with 2 ug of granulocyte-macrophage colony stimulating factor. This indicates
substantially more infiltration of cells from the tissue adjacent the hydrogel (highly
cellular area near the edge of the image) into the pore-forming hydrogel. Figure 5C
and 5D show a comparison of baseline DC invasion into pore-forming hydrogels in which
porogens were formed from alginate dialdehyde (Figure 5C) or from reduced alginate
dialdehyde (Figure 5D) without GM-CSF. Substantially less baseline cell infiltration
occurred absent GM-CSF. For histology, dendritic cells are stained for CD11c (green)
and NIHC-II (red), with Hoescht nuclear counterstain (blue). This figure shows the
difference in host cell recruitment by materials with porogens formed from the oxidized
alginate vs. reduced alginate.
Example 6: Control of Stem Cell Proliferation Within Pore-Forming Hydrogels by Varying
Bulk Phase Composition
[0071] The purpose of this approach is to manipulate cell expansion and release using insoluble
cues. Thus, it was determined whether the density of adhesion ligands and mechanical
properties of the non-degrading hydrogel surrounding porogens would have effects on
the cells. As shown in Figure 6, the density of ligands significantly altered DNA
synthesis, whereas altering elastic modulus altered both DNA synthesis and cell release
over 1 week. Insoluble cues like adhesion ligand density had effects on cells over
long time-frames, as shown by histology in Figure 6D.
[0072] Specifically, studies were performed to determine whether the composition of the
bulk component of pore-forming hydrogels could modulate cell proliferation and engraftment
in vivo. The analysis of 24 hr
3H-thymidine incorporation (proportional to DNA synthesis) by mesenchymal stem cells
(D1; red curve) or cumulative MSC deployment (blue curves) from pore-forming hydrogels
after 7 days of culture as a function of density of RGD peptides in bulk gels with
60 kPa modulus, or elastic modulus of bulk hydrogels presenting 10 RGD peptides /
alginate polymer (data are mean +/- SEM, n = 3-5) is shown in Figure 6A and 6B. RGD
density had significant effects of cell proliferation, whereas elastic modulus had
effects on both proliferation and release (p < 0.05, ANOVA). The analysis of DNA synthesis
as a function of pore formation is shown in Figure 6C. Staining for Ki-67 (proliferation
marker, green) in D1 cells in cryosectioned pore-forming hydrogels after 50 days of
culture is shown in Figure 6D.
Control of deployed stem cell fate via composition of the bulk hydrogel
[0073] Pore forming hydrogels were formed as described in Example 1. By manipulating the
composition (density of integrin-binding RGD peptides and elastic modulus) of the
bulk hydrogel, it was possible to control mesenchymal stem cell (MSC) proliferation
and release
in vitro. In vivo, the overall density of mCherry-labeled mouse MSC deployed into the subcutaneous space
could be increased by increasing the density of RGD peptides from 2 to 10 peptides
per alginate polymer chain (Figure 6E). For therapeutic studies, human MSC were deployed
into Nude Rat cranial defects. After 4 weeks, rats were euthanized, and the degree
of healing (due to new bone formation) was assessed by Hematoxylin/Eosin staining.
Briefly, the area of newly formed bone within the defect was divided by the total
area of the defect to generate the "Percent Healing" metric. Using this quantitative
metric, it was found that delivering MSC in pore-forming hydrogels was substantially
better than delivery via standard hydrogels or saline in terms of ability to induce
new bone formation (Figure 6F). Moreover, the elastic modulus of the bulk hydrogel
component had a substantial effect on new bone formation at 4 weeks, as deployment
from a pore-forming hydrogel with a 60 kPa, 10 RGD / alginate polymer bulk phase led
to significantly more bone formation (
p < 0.05, 2-tailed t-test) than deployment from a pore-forming hydrogel with an 8 kPa,
10 RGD / alginate polymer bulk phase.
[0074] Thus, when mCherry-labeled D1 were deployed into the subcutaneous tissues of Nude
mice via pore-forming hydrogels, increasing the RGD density of the bulk component
from 2 to 10 RGD peptides / alginate polymer substantially increased the overall number
of engrafted cells without significantly affecting cell deployment kinetics.
[0075] Though the example here demonstrated an effect of bulk hydrogel elasticity on cell-mediated
tissue regeneration, as described herein, many other aspects of the bulk hydrogel
phase - for example, the presentation of matrix-bound growth factors or peptide-mimics
thereof - are engineered to influence cell-mediated tissue regeneration.
Example 7: Mechanical properties and in-vitro degradation of hydrogels formed from
binary alginates
[0076] Elastic moduli and degradation of bulk hydrogels formed by cross-linking binary combinations
of oxidized alginate (5% theoretical degree of oxidation) at a constant density of
20 mg/mL with unmodified, high M
w alginate are shown in Figure 7A and Figure 7B. Degradation was assessed by comparing
the dry mass after 4 days
in-vitro to initial dry mass. The diameters of porogens formed from binary mixtures of 20
mg/mL oxidized alginate with 7.5 mg/mL unmodified alginate is shown in Figure 7C.
Porogen diameter was measured by processing fluorescent micrographs of porogens prepared
from aminofluorescenin-labeled alginates.
[0077] The patent and scientific literature referred to herein establishes the knowledge
that is available to those with skill in the art.
1. A composition comprising a porogen hydrogel and a bulk hydrogel, which composition
is not initially macroporous and becomes macroporous over time when resident in the
body of a recipient, wherein said porogen hydrogel degrades at least 10% faster than
said bulk hydrogel following residence in said subject, leaving macropores having
a diameter of greater than 20 µm in its place, and wherein
(a) said porogen hydrogel comprises oxidized alginate; or
(b) said porogen hydrogel comprises a shorter polymer than said bulk hydrogel.
2. The composition of claim 1, wherein said porogen hydrogel or said bulk hydrogel or
both hydrogels comprise a bioactive factor.
3. The composition of claim 1, wherein said porogen hydrogel or said bulk hydrogel or
both hydrogels recruit a cell into the composition.
4. The composition of claim 1, wherein said porogen hydrogel or said bulk hydrogel comprises
an isolated cell.
5. The composition of claim 4, wherein said bulk hydrogel is cross-linked around said
porogen hydrogel or wherein said porogen hydrogel is physically entrapped in said
bulk hydrogel.
6. The composition of claim 1, comprising a bioactive factor selected from the group
consisting of vascular endothelial growth factor (VEGF), acidic fibroblast growth
factor (aFGF), basic fibroblast growth factor (bFGF), placenta growth factor (PIGF),
platelet derived growth factor (PDGF), leptin, hematopoietic growth factor (HGF),
VEGF receptor-1 (VEGFR-1), VEGFR-2, a member of the bone morphogenetic protein (BMP)
family, granulocyte/macrophage colony stimulating factor (GM-CSF), FMS-like tyrosine
kinase 3 ligand (Flt3 ligand), hepatocyte growth factor, stromal derived factor 1
(SDF-1), insulin like growth factor (IGF), anti-VEGF antibody, anti-aFGF antibody,
anti-bFGF antibody, anti-PIGF antibody, anti-leptin antibody, anti-HGF antibody, anti-VEGFR-1
antibody, anti-VEGFR-2 antibody, anti-PDGF antibody, anti-BMP antibody, anti-Flt3
ligand, and anti-IGF antibody.
7. The composition of claim 4, wherein said cell is a mesenchymal stem cell, a myoblast,
a vascular progenitor cell, a differentiated cell derived from an embryonic stem cell
or an induced pluripotent stem cell, an induced pluripotent cell, or a cell that was
directly reprogrammed from a fibroblast to a differentiated state.
8. The composition of claim 1, wherein said porogen hydrogel comprises an elastic modulus
of between 20 kPa and 60 kPa.
9. The composition of claim 1, wherein said bulk hydrogel
(a) comprises a peptide comprising an amino acid sequence of PHSRN, DGEA, or RGD;
or
(b) comprises Adensity of RGD peptides from 2 to 10 peptides per alginate polymer
chain; or
(c) comprises an initial elastic modulus of at least 40 kPa.
10. The composition of claim 1 or 2, wherein said porogen hydrogel or said bulk hydrogel
comprises a bioactive factor for use in promoting bone or cartilage repair, regeneration,
or formation, wherein said bioactive factor optionally comprises BMP-2, BMP-4, or
RunX.
11. The composition of claim 10, wherein said porogen hydrogel or said bulk hydrogel
(a) comprises an isolated bone cell selected from the group consisting of an osteoblast,
an osteocyte, an osteoclast, and an osteoprogenitor; or
(b) comprises an isolated cartilage cell, wherein said isolated cartilage cell comprises
a chondroblast; and wherein said isolated bone cell is optionally an autologous or
allogenic cell.
12. The composition of claim 1 or 2, wherein said porogen hydrogel or said bulk hydrogel
comprises a bioactive factor for use in muscle repair, regeneration, or formation.
13. The composition of claim 12, wherein
(a) said bioactive factor comprises MyoD; or
(b) said porogen hydrogel or said bulk hydrogel comprises an isolated muscle cell
selected from the group consisting of a skeletal muscle cell, a cardiac muscle cell,
a smooth muscle cell, and a myo-progenitor cell, wherein said isolated muscle cell
is optionally an autologous or allogenic cell.
14. The composition of claim 1 or 2, wherein said porogen hydrogel or said bulk hydrogel
comprises a bioactive factor for use in skin repair, regeneration, or formation, wherein
said bioactive factor optionally comprises FGF.
15. The composition of claim 14, wherein said porogen hydrogel or said bulk hydrogel comprises
an isolated skin cell selected from the group consisting of a fibroblast, a dermal
cell, an epidermal cell, or a dermal progenitor cell, wherein said isolated skin cell
is optionally an autologous cell or an allogeneic cell.
16. The composition of claim 1, further comprising an isolated cell, for deploying cells
from a scaffold into tissues of a mammalian subject, wherein said composition lacks
macropores at the time of administration, and wherein said composition comprises macropores
following residence in said subject.
17. The composition of claim 1, for recruiting cells into a scaffold in vivo, wherein said composition lacks macropores at the time of administration, and wherein
said composition comprises macropores following residence in said subject.
18. The composition of claim 1, wherein the porogen hydrogel and/or the bulk hydrogel
comprises a cross-linked alginate polymer comprising a divalent cation selected from
the group consisting of Ca+2, Mg+2, and Ba+2.
19. The composition of claim 1, wherein the porogen hydrogel comprises oxidized alginate,
and wherein the porogen hydrogel is more hydrolytically degradable than the bulk hydrogel.
20. The composition of claim 19, wherein more than 5% of the sugar residues in the alginate
are oxidized.
21. The composition of claim 1, wherein said porogen hydrogel comprises a shorter polymer
than said bulk hydrogel.
22. The composition of claim 1, wherein the porogen hydrogel comprises an oxidized alginate
polymer having a molecular weight from 5,000 to 500,000 Daltons (Da).
23. The composition of claim 22, wherein the bulk hydrogel comprises an alginate polymer
having a molecular weight from 5,000 to 500,000 Da.
1. Zusammensetzung, welche ein porogenes Hydrogel und ein Bulk-Hydrogel umfasst, worin
die Zusammensetzung ursprünglich nicht makroporig ist und über die Zeit makroporig
wird, wenn sie in dem Körper eines Empfängers verbleibt, wobei das porogene Hydrogel
nach Aufnahme in das Individuum mindestens 10% schneller als das Bulk-Hydrogel abgebaut
wird, wobei es Makroporen mit einem Durchmesser von mehr als 20 µm an seiner Stelle
hinterlässt, und worin
(a) das porogene Hydrogel oxidiertes Alginat umfasst; oder
(b) das porogene Hydrogel ein kürzeres Polymer als das Bulk-Hydrogel umfasst.
2. Zusammensetzung nach Anspruch 1, worin das porogene Hydrogel oder das Bulk-Hydrogel
oder beide Hydrogele einen bioaktiven Faktor umfassen.
3. Zusammensetzung nach Anspruch 1, worin das porogene Hydrogel oder das Bulk-Hydrogel
oder beide Hydrogele eine Zelle in die Zusammensetzung einschleusen.
4. Zusammensetzung nach Anspruch 1, worin das porogene Hydrogel oder das Bulk-Hydrogel
eine isolierte Zelle umfassen.
5. Zusammensetzung nach Anspruch 4, worin das Bulk-Hydrogel um das porogene Hydrogel
vernetzt ist oder worin das porogene Hydrogel in dem Bulk-Hydrogel körperlich gefangen
ist.
6. Zusammensetzung nach Anspruch 1, welche einen bioaktiven Faktor umfasst, ausgewählt
aus der Gruppe bestehend aus vaskularem endothelialem Wachstumsfaktor (VEGF), saurem
Fibroblasten-Wachstumsfaktor (aFGF), basischem Fibroblasten-Wachstumsfaktor (bFGF),
Plazenta-Wachstumsfaktor (PIGF), Plättchen-abgeleitetem Wachstumsfaktor (PDGF), Leptin,
hämatopoetischem Wachstumsfaktor (HGF), VEGF Rezeptor-1 (VEGFR-I), VEGFR-2, einem
Mitglied der knochenmorphogenetischen Protein- (BMP) Familie, Granulozyten/Makrophagen-Kolonie-stimulierendem
Faktor (GM-CSF), FMS-ähnliche Tyrosinkinase-3-Ligand (Flt3-Ligand), hepatozytärem
Wachstumsfaktor, Stroma-abgeleitetem Faktor 1 (SDF-1), insulinähnlichem Wachstumsfaktor
(IGF), Anti-VEGF-Antikörper, Anti-aFGF-Antikörper, Anti-bFGF-Antikörper, Anti-PIGF-Antikörper,
Anti-Leptin-Antikörper, Anti-HGF-Antikörper, Anti-VEGFR-1-Antikörper, Anti-VEGFR-2-Antikörper,
Anti-PDGF-Antikörper, Anti-BMP-Antikörper, Anti-Flt3-Ligand, und Anti-IGF-Antikörper.
7. Zusammensetzung nach Anspruch 4, worin die Zelle eine mesenchymale Stammzelle, ein
Myoblast, eine vaskuläre Vorläuferzelle, eine von einer embryonalen Stammzelle abgeleitete
differenzierte Zelle oder eine induzierte pluripotente Stammzelle, eine induzierte
pluripotente Zelle, oder eine Zelle ist, die von einem Fibroblasten direkt in ein
differenziertes Stadium umprogrammiert wurde.
8. Zusammensetzung nach Anspruch 1, worin das porogene Hydrogel ein Elastizitätsmodul
von zwischen 20 kPa und 60 kPa umfasst.
9. Zusammensetzung nach Anspruch 1, worin das Bulk-Hydrogel
(a) ein Peptid umfasst, das eine Aminosäure-Sequenz von PHSRN, DGEA, oder RGD umfasst;
oder
(b) eine Dichte von RGD-Peptiden von 2 bis 10 Peptiden pro Alginat-Polymer-Kette umfasst;
oder
(c) einen initialen Elastizitätsmodul von mindestens 40 kPa umfasst.
10. Zusammensetzung nach Anspruch 1 oder 2, worin das porogene Hydrogel oder das Bulk-Hydrogel
einen bioaktiven Faktor umfasst, zur Verwendung bei dem Fördern von Knochen oder Knorpelheilung,
Regeneration oder Bildung, worin der bioaktive Faktor wahlweise BMP-2, BMP-4, oder
RunX umfasst.
11. Zusammensetzung nach Anspruch 10, worin das porogene Hydrogel oder das Bulk-Hydrogel
(a) eine isolierte Knochenzelle umfasst, ausgewählt aus der Gruppe bestehend aus einem
Osteoblasten, einer Osteozyte, einem Osteoklasten, und einer Knochenvorläuferzelle;
oder
(b) eine isolierte Knorpelzelle umfasst, worin die isolierte Knorpelzelle einen Chondroblasten
umfasst; und worin die isolierte Knochenzelle wahlweise eine autologe oder allogene
Zelle ist.
12. Zusammensetzung nach Anspruch 1 oder 2, worin das porogene Hydrogel oder das Bulk-Hydrogel
einen bioaktiven Faktor zur Verwendung bei Muskelheilung, Regeneration, oder Ausbildung
umfasst.
13. Zusammensetzung nach Anspruch 12, worin
(a) der bioaktive Faktor MyoD umfasst; oder
(b) das porogene Hydrogel oder das Bulk-Hydrogel eine isolierte Muskelzelle umfasst,
ausgewählt aus der Gruppe bestehend aus einer Skelettmuskelzelle, einer Herzmuskelzelle,
einer glatten Muskelzelle, und einer Muskelvorläuferzelle, worin die isolierte Muskelzelle
wahlweise eine autologe oder allogene Zelle ist.
14. Zusammensetzung nach Anspruch 1 oder 2, worin das porogene Hydrogel oder das Bulk-Hydrogel
einen bioaktiven Faktor umfasst, zur Verwendung bei Hautheilung, Regeneration, oder
Ausbildung, worin der bioaktive Faktor wahlweise FGF umfasst.
15. Zusammensetzung nach Anspruch 14, worin das porogenes Hydrogel oder das Bulk-Hydrogel
eine isolierte Hautzelle umfasst, ausgewählt aus der Gruppe bestehend aus einem Fibroblasten,
einer Hautzelle, einer Epidermiszelle, oder einer Hautvorläuferzelle, worin die isolierte
Hautzelle wahlweise ein autologe Zelle oder eine allogene Zelle ist.
16. Zusammensetzung nach Anspruch 1, welche weiter eine isolierte Zelle zum Einschleusen
von Zellen aus einem Gerüst in Gewebe eines Säugers umfasst, worin die Zusammensetzung
zu der Zeit der Verabreichung keine Makroporen aufweist, und worin die Zusammensetzung
nach Aufnahme in das Individuum Makroporen umfasst.
17. Zusammensetzung nach Anspruch 1, zum Einschleusen von Zellen in ein Gerüst in vivo, worin die Zusammensetzung zu der Zeit der Verabreichung keine Makroporen aufweist,
und worin die Zusammensetzung nach Aufnahme in das Individuum Makroporen umfasst.
18. Zusammensetzung nach Anspruch 1, worin das porogene Hydrogel und/oder das Bulk-Hydrogel
ein quervernetztes Alginat-Polymer umfasst, das ein divalentes Kation umfasst, ausgewählt
aus der Gruppe bestehend aus Ca+2, Mg+2, und Ba+2.
19. Zusammensetzung nach Anspruch 1, worin das porogene Hydrogel oxidiertes Alginat umfasst,
und worin das porogene Hydrogel hydrolytisch abbaubarer ist, als das Bulk-Hydrogel.
20. Zusammensetzung nach Anspruch 19, worin mehr als 5% der Zuckerreste in dem Alginat
oxidiert sind.
21. Zusammensetzung nach Anspruch 1, worin das porogene Hydrogel ein kürzeres Polymer
umfasst als das Bulk-Hydrogel.
22. Zusammensetzung nach Anspruch 1, worin das porogene Hydrogel ein oxidiertes Alginat-Polymer
mit einem Molekulargewicht von 5.000 bis 500.000 Dalton (Da) umfasst.
23. Zusammensetzung nach Anspruch 22, worin das Bulk-Hydrogel ein Alginat-Polymer mit
einem Molekulargewicht von 5.000 bis 500.000 Da umfasst.
1. Composition comprenant un hydrogel porogène et un hydrogel massif, laquelle composition
n'est pas initialement macroporeuse et devient macroporeuse au cours du temps lors
du séjour dans le corps d'un receveur, ledit hydrogel porogène se dégradant au moins
10% plus vite que ledit hydrogel massif à la suite du séjour dans ledit sujet, laissant
des macropores ayant un diamètre de plus de 20 µm à sa place, et dans laquelle :
(a) ledit hydrogel porogène comprend de l'alginate oxydé ; ou
(b) ledit hydrogel porogène comprend un polymère plus court que ledit hydrogel massif.
2. Composition selon la revendication 1, dans laquelle ledit hydrogel porogène ou ledit
hydrogel massif ou les deux hydrogels comprennent un facteur bioactif.
3. Composition selon la revendication 1, dans laquelle ledit hydrogel porogène ou ledit
hydrogel massif ou les deux hydrogels recrutent une cellule dans la composition.
4. Composition selon la revendication 1, dans laquelle ledit hydrogel porogène ou ledit
hydrogel massif comprend une cellule isolée.
5. Composition selon la revendication 4, dans laquelle ledit hydrogel massif est réticulé
autour dudit hydrogel porogène ou dans laquelle ledit hydrogel porogène est physiquement
piégé dans ledit hydrogel massif.
6. Composition selon la revendication 1, comprenant un facteur bioactif choisi dans le
groupe consistant en le facteur de croissance de l'endothélium vasculaire (VEGF),
le facteur de croissance acide des fibroblastes (aFGF), le facteur de croissance basique
des fibroblastes (bFGF), le facteur de croissance placentaire (PIGF), le facteur de
croissance dérivé des plaquettes (PDGF), la leptine, le facteur de croissance hématopoïétique
(HGF), le récepteur de VEGF 1 (VEGFR-1), le VEGFR-2, un membre de la famille des protéines
morphogénétiques osseuses (BMP), le facteur de stimulation de la formation de colonies
de granulocytes/macrophages (GM-CSF), le ligand tyrosine kinase 3 de type FMS (ligand
Flt3), le facteur de croissance des hépatocytes, le facteur 1 dérivé des cellules
stromales (SDF-1), le facteur de croissance insulinoïde (IGF), l'anticorps anti-VEGF,
l'anticorps anti-aFGF, l'anticorps anti-bFGF, l'anticorps anti-PIGF, l'anticorps anti-leptine,
l'anticorps anti-HGF, l'anticorps anti-VEGFR-1, l'anticorps anti-VEGFR-2, l'anticorps
anti-PDGF, l'anticorps anti-BMP, l'anti-ligand Flt3 et l'anticorps anti-IGF.
7. Composition selon la revendication 4, dans laquelle ladite cellule est une cellule
souche mésenchymateuse, un myoblaste, une cellule progénitrice vasculaire, une cellule
différenciée dérivée d'une cellule souche embryonnaire ou d'une cellule souche pluripotente
induite, une cellule pluripotente induite ou une cellule qui a été directement reprogrammée
à partir d'un fibroblaste vers un état différencié.
8. Composition selon la revendication 1, dans laquelle ledit hydrogel porogène comprend
un module élastique d'entre 20 kPa et 60 kPa.
9. Composition selon la revendication 1, dans laquelle ledit hydrogel massif
(a) comprend un peptide comprenant une séquence d'acides aminés de PHSRN, DGEA ou
RGD ; ou
(b) comprend une densité de peptides RGD de 2 à 10 peptides par chaîne de polymère
d'alginate ; ou
(c) comprend un module élastique initial d'au moins 40 kPa.
10. Composition selon l'une des revendications 1 et 2, dans laquelle ledit hydrogel porogène
ou ledit hydrogel massif comprend un facteur bioactif pour une utilisation pour favoriser
la réparation, la régénération ou la formation osseuse ou cartilagineuse, ledit facteur
bioactif comprenant facultativement BMP-2, BMP-4 ou RunX.
11. Composition selon la revendication 10, dans laquelle ledit hydrogel porogène ou ledit
hydrogel massif
(a) comprend une cellule osseuse isolée choisie dans le groupe consistant en un ostéoblaste,
un ostéocyte, un ostéoclaste et une cellule ostéoprogénitrice ; ou
(b) comprend une cellule cartilagineuse isolée, ladite cellule cartilagineuse isolée
comprenant un chondroblaste ; et ladite cellule osseuse isolée étant facultativement
une cellule autologue ou allogénique.
12. Composition selon l'une des revendications 1 et 2, dans laquelle ledit hydrogel porogène
ou ledit hydrogel massif comprend un facteur bioactif pour une utilisation dans la
réparation, la régénération ou la formation musculaire.
13. Composition selon la revendication 12, dans laquelle :
(a) ledit facteur bioactif comprend MyoD ; ou
(b) ledit hydrogel porogène ou ledit hydrogel massif comprend une cellule de muscle
isolée choisie dans le groupe consistant en une cellule de muscle squelettique, une
cellule de muscle cardiaque, une cellule de muscle lisse et une cellule myo-progénitrice,
ladite cellule de muscle isolée étant facultativement une cellule autologue ou allogénique.
14. Composition selon l'une des revendications 1 et 2, dans laquelle ledit hydrogel porogène
ou ledit hydrogel massif comprend un facteur bioactif pour une utilisation dans la
réparation, la régénération ou la formation de la peau, ledit facteur bioactif comprenant
facultativement FGF.
15. Composition selon la revendication 14, dans laquelle ledit hydrogel porogène ou ledit
hydrogel massif comprend une cellule de peau isolée choisie dans le groupe consistant
en un fibroblaste, une cellule du derme, une cellule épidermique ou une cellule progénitrice
du derme ladite cellule de peau isolée étant facultativement une cellule autologue
ou allogénique.
16. Composition selon la revendication 1, comprenant en outre une cellule isolée, pour
déployer des cellules à partir d'un échafaudage dans des tissus d'un sujet mammifère,
ladite composition étant dépourvue de macropores au moment de l'administration, et
ladite composition comprenant des macropores à la suite du séjour dans ledit sujet.
17. Composition selon la revendication 1, pour le recrutement de cellules dans un échafaudage
in vivo, ladite composition étant dépourvue de macropores au moment de l'administration, et
ladite composition comprenant des macropores à la suite du séjour dans ledit sujet.
18. Composition selon la revendication 1, dans laquelle l'hydrogel porogène et/ou l'hydrogel
massif comprennent un polymère d'alginate réticulé comprenant un cation divalent choisi
dans le groupe consistant en Ca2+, Mg2+ et Ba2+.
19. Composition selon la revendication 1, dans laquelle l'hydrogel porogène comprend un
alginate oxydé, et dans laquelle l'hydrogel porogène est davantage hydrolytiquement
dégradable que l'hydrogel massif.
20. Composition selon la revendication 19, dans laquelle plus de 5 % des résidus de sucre
dans l'alginate sont oxydés.
21. Composition selon la revendication 1, dans laquelle ledit hydrogel porogène comprend
un polymère plus court que ledit hydrogel massif.
22. Composition selon la revendication 1, dans laquelle l'hydrogel porogène comprend un
polymère d'alginate oxydé ayant une masse moléculaire de 5000 à 500 000 Daltons (Da).
23. Composition selon la revendication 22, dans laquelle l'hydrogel massif comprend un
polymère d'alginate ayant une masse moléculaire de 5000 à 500 000 Da.